This invention is generally in the field of protein or peptide mapping, and more particularly relates to mapping that utilizes hydrogen/deuterium exchange.
Proteins contain covalently bonded hydrogen atoms that are known to exchange with hydrogen atoms found in a surrounding solvent. Thus, if the solvent surrounding a protein is changed from normal water (H2O) to heavy water (D2O), the exchange of hydrogen (1H) for deuterium (2H) can be observed. This exchange reaction, known as hydrogen-deuterium exchange (HDX), increases the molecular mass of the protein because the deuterium nucleus is heavier than the common proton. Since the hydrogen-deuterium exchange rate reflects the exposure level of the particular hydrogen atom to the solvent, spectrometric analysis can be utilized subsequent to HDX to determine which portions of the protein have increased in molecular mass and thus are accessible to the surrounding solvent. This information can be extremely useful in understanding protein conformation, protein/protein interactions, and protein/ligand interactions. See Wang, et al., Biochemistry 37:15289-99 (1998). For example, this “mapping” information may be used to determine protein-ligand binding sites and is used in the pharmaceutical industry for intelligent drug design.
Determining which portions of a protein have increased in molecular mass due to HDX (and thus which portions of a protein are in contact with the surrounding solvent) generally requires a protein digestion step, a desalting/separation step, and a spectrometric analysis step. Currently, the separation technology used in HDX experiments is reversed phase high performance liquid chromatography (RP-HPLC). Unfortunately, the mobile phase used in RP-HPLC is predominately water. The presence of water is a major drawback because it allows for the back-exchange of deuterium for hydrogen during the separation step immediately prior to the spectrometric analysis. Back-exchange is a major problem with solution HDX, as the back-exchange undesirably reduces both the amount and the resolution of the resulting data. It would therefore be useful to provide a method that eliminates, or at least substantially reduces, back-exchange during separation.
In order to limit back-exchange, RP-HPLC conventionally is performed at 0-4° C. and under very fast gradients in order to limit the period in which the sample is in contact with the aqueous phase. Unfortunately, this separation method reduces the reversed-phase chromatographic resolution, and increases the complexity of the spectra. Although reduced chromatographic resolution can be partially offset through the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS), these techniques still allow for the back exchange of deuterium for hydrogen. It therefore would be desirable to eliminate, or at least substantially reduce, back-exchange during separation while maintaining a high degree of chromatographic resolution.